Although heterosexual transmission is the most important manner by which HIV is spread throughout the world today, very little is known about the mechanisms involved in the initiation and establishment of infection at the mucosal surfaces of the genital tract. The goals of this project are to determine if specific genotypes of SIV are preferentially transmissible by the mucosal route, to develop in vitro culture systems which will facilitate investigation of the molecular and cellular determinants of mucosal infection, and to investigate the role of immune cells of the cervicovaginal mucosa in the initial stages of infection with SIV in rhesus macaques. We are conducting a pilot study designed to work out the mechanics of intravaginal inoculation and compare infection efficiency between lymphotropic SIVmac239 and macrophage tropic SIVmac239/17E. For these experiments, we are using cell-free virus stocks which have been derived from and titrated in rhesus PBMC. Single intravaginal inoculations using 104 TCID50 of either SIVmac239 (n=3) or SIVmac239/17E (n=3) failed to infect any animals. One animal from each virus group from the first round of inoculations was then re-inoculated on two consecutive days with 105 TCID50 (2 x 105 TCID50 total dose). Of these two macaques, only the animal inoculated with macrophage tropic virus has become infected. We have now re-inoculated the remaining uninfected macaques from the pilot study, using two inoculations (2.5 x 104 TCID50 per inoculation) on consecutive days to test the repeatability of this system using a reduced dose of virus. After we determine the infection efficiencies for these two viruses, we will clone and analyze the envelope genes derived from mucosally-infected macaques and compare their sequences and biological abilities in vitro. Vaginal and endocervical epithelial cells which line the female genital tract represent the first mucosal barrier of defense against heterosexual transmission of HIV-1. As a model system to study the mechanism of HIV-1/SIV traversal of the vaginal and endocervical mucosa, we have developed isolation procedures for the establishment and maintenance of primary cultures of both vaginal and endocervical tissues. Using these cultures, we hope to mimic in vitro the mucosal barrier maintained at these tissue sites in vivo. Both stromal (fibroblastic) and epithelial cell-enriched cultures are currently being maintained and passaged in our laboratory. The cultures retain viability for up to 6 passages, or approximately 2 months. Work in progress includes viral binding studies with these cultures as well as infectivity assays. We have also had encouraging results growing these cultures in a multilayered fashion on permeable membrane supports. To further enhance our in vitro model of the muco sa of the lower reproductive tract of rhesus macaques we are now isolating dendritic cells from rhesus tonsillar explants which will be added to the lower (stromal) chamber of the filter apparatus. In order to identify potential target cells for infection with SIV, we are using an immunohistochemistry assay to investigate the phenotype, number, and distribution pattern of immune cells in tissue sections of cervico-vaginal mucosa from mature SIV negative rhesus macaques. Our future plans include identification of the initial target cells of infection following intravaginal inoculation with macrophage or lymphocyte tropic SIV variants, using in situ PCR and in situ RT-PCR techniques which we are currently developing. We also plan to examine the expression of various adhesion and activation molecules in sections of vagina and cervix from both uninfected and acutely infected rhesus, as well as evaluate the mucosal immune response in situ following mucosal SIV infection through the detection of mRNA of various cytokines (IL-2, IL-4, IL-6, IL-10, and IFN-g) by in situ hybridization. For these experiments we will use digoxigenin-labeled riboprobes constructed using a protocol develop ed by Dr. R. P. Bucy and plasmid templates provided by Dr. Fran[unreadable]ois Villinger.